Despite significant improvements in living standards and biomedical technologies over the past century, the global burden of infectious diseases remains exceedingly high and is a major cause of public health, economic and social problems. According to World Health Organization (WHO) statistics, infectious and parasitic diseases such as pneumonia, tuberculosis, meningitis, diarrheal diseases, HIV and malaria are the second leading causes of death worldwide. The widespread and often indiscriminate use of antibiotics in industrialized nations further fuels the problem by contributing to the rapid emergence of drug resistant pathogens, making infectious diseases increasingly difficult to control with the existing classes of antibiotics. The exploding crisis of antibiotic-resistant infections coupled with the on-going dearth in new small-molecule antibiotics development, have spurred considerable efforts toward the discovery and development of membrane active antimicrobial peptides (AMPs) as an alternative class of antimicrobial agents. Naturally occurring antimicrobial peptides, also known as ‘host defense peptides’, were first discovered as components of the innate immunity, forming the first line of defense against invading pathogens in all living organisms. Unlike conventional antibiotics that inhibit specific biosynthetic pathways such as cell wall or protein synthesis, the majority of the cationic antimicrobial peptides exert their activities via physical disruption of the more negatively charged microbial membrane lipid bilayers to induce leakage of cytoplasmic components leading to cell death. The physical nature of membrane disruption is believed to result in a lower likelihood for drug resistance development as it becomes metabolically ‘costlier’ for the microorganism to mutate or to repair its membrane components at the same rate as the damage is being inflicted.
Although more than 1700 naturally occurring antimicrobial peptides from diverse sources including microorganisms, plants and animals have been isolated and characterized in the past 3 decades, only very few AMPs such as polymyxins and gramicidins are being used clinically; and mainly in topical formulations due to their high systemic toxicities. The major challenges identified with the application of antimicrobial peptides as drugs lie in the high cost in synthesizing long peptide sequences, poor stability and unknown toxicity after systemic administration. In efforts to enhance antimicrobial activities and minimize non-specific toxicities, more researchers are increasingly utilizing naturally occurring antimicrobial peptide or protein sequences as templates to perform chemical modifications such as cyclization, sequence truncations, and substitution with D-, β- or fluorinated-amino acids for the generation of new peptide analogs with broader applications for localized or systemic infections within the body. However, current approaches to optimize naturally occurring antimicrobial peptide sequences remain largely empirical at best, making it extremely difficult to delineate general structure-activity relationships especially against the backdrop of massive sequence and structural diversities. Furthermore, many of the new peptide analogs remain long (20 amino acids or more), which might induce significant immunogenicity and ultimately increase the cost for large scale manufacturing. More importantly, it has been suggested that the use of antimicrobial peptides with sequences that are too close to the host defense antimicrobial peptides may trigger the development of resistance towards innate AMPs that could inevitably compromise natural defenses against infections, posing significant health and environmental risks.
At the same time, the rapid emergence of antibiotics resistant bacteria and fungi in both the nosocomial and community settings has created a significant strain on healthcare systems around the world. While global incidences of antibiotics resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and multidrug-resistant Klebsiella pneumoniae and Acinetobacter spp. have reached epidemic levels, the number of new antibiotics entering the clinical development pipeline has been dismal; with only three new structural classes of antibiotics including the oxazolidinones (linezolid), lipopeptides (daptomycin) and pleuromutilins (retapamulin) entering the market since 2000. This development is especially alarming given that pathogenic bacteria such as S. aureus, Enterobacter and Klebsiella are developing resistance to vancomycin and carbapenems, which are potent antibiotics traditionally reserved as the last line of defense for vulnerable patients in hospitals. With the on-going dearth in small molecular antibiotics development, the design and identification of alternative classes of antimicrobial agents with new modes of action that can effectively overcome drug resistance mechanisms is more pressing than ever.
As the majority of the antimicrobial peptides exert their antimicrobial activities through a rapid and direct membrane lytic mechanism, they possess an inherent advantage in overcoming conventional mechanisms of antibiotics resistance such as the increased expression of drug efflux pumps on microbial membranes, production of drug degradation enzymes or alteration to drug interaction sites acquired by microbes against small molecular antibiotics targeting specific biosynthetic pathways. Significant barriers limiting the successful clinical translation of antimicrobial peptides, however, include high systemic toxicities as a result of poor microbial membrane selectivities, relatively high manufacturing cost (for long peptide sequences) and susceptibility to degradation by proteases present in biological fluids such as blood serum, wound exudates or lacrimal fluids.
Keratitis continues to be an important cause of ocular morbidity, and it is also a major eye disease that leads to blindness. Symptoms of keratitis include pain and redness in the eye, blurred vision, sensitivity to light, and excessive tearing or discharge. It may lead to severe vision loss and scars on the cornea. Moreover, the incidence of keratitis has increased over the past 30 years mainly due to the frequent use of topical corticosteroids and antibacterial agents in the treatment of patients with keratitis, as well as the rise in the number of patients who are immunocompromised.
Causes of keratitis include herpes simplex virus type 1, varicella zoster, and adenoviruses, bacteria, parasites, fungi and vitamin A deficiency. An outbreak of keratitis was reported in 2005 and 2006 in the United States and also in France, Hong Kong and Singapore. Most of the cases in the United States involved soft contact lens wear, which was mainly caused by fungal infections.
Keratitis remains a diagnostic and therapeutic challenge to the ophthalmologist due to several reasons. The main challenge is fast and accurate diagnosis. Diagnosis of keratitis is typically conducted by culture or corneal biopsy. However, isolating and identifying the species of organism in the laboratory take time, leading to late diagnosis frequently. Furthermore, as keratitis can be caused by various microorganisms, the different types of keratitis are often misdiagnosed as one another. For example, fungal keratitis is often misdiagnosed as bacterial keratitis as clinicians often consider fungal keratitis only after a presumed bacterial keratitis worsens during antibiotic therapy. Moreover, anti-fungal sensitivity testing is unreliable, and correlates poorly with clinical efficacy. Even if the diagnosis is made accurately, management of the condition remains a challenge because of the poor corneal penetration and the limited commercial availability of agents useful in treating keratitis.
Most commercially available medications are merely fungistatic or bactericidal, and they require an intact immune system and prolonged therapeutic course for successful resolution of the condition. Such microbe specific medications are disadvantages as it requires specific diagnosis of the cause of keratitis before treatment can be provided. Furthermore, commercially available anti-fungal medication does not address the fact that fungal keratitis infection often exists as a biofilm, which is particularly difficult to remove because fungal cells are encapsulated in a protective and impermeable extracellular matrix (ECM). Therefore, typically, treatment requires significantly higher doses of antifungal agents in an attempt to clear biofilm.
Furthermore, current commercial drugs include azole compounds (such as funconazole) and polyenes (such as amphopterin B, nystatin and natamycin). The azoles are fungistatic, which function by enzyme inhibition and are prone to drug resistance development. However, azoles available in the art are extremely unstable; their topical solution must be kept refrigerated for no more than 48 hours and protected from light. Another antifungal drugs known in the art are the polyenes, which function via disrupting the permeability of ions through the cell membrane, are rather expensive, poorly water soluble and quite unstable in aqueous, acid or alkaline media, or when exposed to light and excessive heat. All these limit their clinical applications.
In view of the above, there is a need to provide alternative agents useful for treating keratitis caused by various microbes. There is also a need to provide alternative agents useful for treating keratitis that is stable and safe to use.